The formation of tobacco-specific N-nitrosamines (TSNA) during leaf storage is substantial and is influenced by factors such as temperature, leaf moisture and nitrate content. The controlled experiments were conducted in this study to investigate the interaction relationship and effect on TSNA formation during leaf storage in incubation chambers. Leaf moisture was set at three levels (7.71%, 15.30% and 22.10%), which were achieved by balancing leaf moisture with solutions of potassium carbonate of different saturation degrees in enclosed cubes. Leaf samples with different moistures were then stored at five different storage temperatures (10 °C, 20 °C, 30 °C, 40 °C and 50 °C) for 15 days. The results showed that both storage temperature and leaf moisture content had significant effects on TSNA formation and they had significant interaction effects. Total TSNA content of low moisture tobacco (7.71%) increased from 0.8 µg/g to 8.9 µg/g as storage temperature increased from 10 °C to 50 °C, while that of high moisture tobacco (22.10%) increased from 1.7 µg/g to 2.2 µg/g within the same temperature range. Analysis showed that storage temperature was the principle contributor to total TSNA variation with the contribution percentage of 56.1%, followed by leaf moisture content (25.6%) and interaction effect (18.2%). The two factor models were established to reflect the response of individual and total TSNA formation to storage temperature and leaf moisture with the determination coefficients of 96.0%, 95.5%, 96.5%, 93.2% and 96.1% for NAT, NNN, NAB, NNK and total TSNA, respectively. Based on the results and analysis, it was suggested that storage temperature be controlled lower than 25.5 °C and leaf moisture be at moderately higher level as long as not exceeding 18% so as to effectively inhibit the TSNA formation of storing tobacco leaves.